Regulation of apoptosis in aquatic organisms by aquabirnavirus

ABSTRACT

The present invention provides a mechanism for studies of apoptosis in aquatic organisms by infecting the aquatic organisms with aquabimavirus, especially infectious pancreatic necrosis virus (IPNV). The infection of IPNV in an aquatic cell such as a Chinook salmon embryo cell (CHSE-214) converts the cell into an apoptotic cell. The present invention also provides a method for monitoring the morphological changes during apoptosis by cloning EGFP (a variant type of GFP) to an aquatic cell and monitoring the fluorescence using microscopic techniques. The intensity of the fluorescence reflects the expression of EGFP in cells. Finally, the present invention provides means for inducing or preventing/rescuing apoptosis in a host, which include aquatic and vertebrate. The apoptosis can be induced by IPNV infection or VP3 transfection.that VP3 is a 32 kDa protein derived from IPNV segment A. The apoptosis can be prevented or rescued by an antisense VP3 RNA or a zfMcl-1a protein.

RELATED INVENTION

[0001] This patent application claims the priority of U.S. applicationSer. No. 09/706,869, filed Nov. 7, 2000 and U.S. provisional applicationserial No. 60/167,010, filed on Nov. 23, 1999, which are incorporatedherein by reference.

FIELD OF THE INVENTION

[0002] The present invention, certain aspects of which are published inVirus Research (1999), 63:75-83 and J. Virol. (1999), 73:5056-5063(which are herein incorporated by reference), relates to apoptosis inaquatic organisms induced by aquabimavirus, preferably infectiouspancreatic necrosis virus (IPNV). It also relates to methods to regulateapoptosis in aquatic cells by controlling the expression of Mcl-1 geneand pre-treating the aquatic cells with drugs which block the viralreplication. It further relates to a method for monitoring the progressof apoptosis using EGFP (a variant type of green fluorescent protein[GFP]) as a probe.

BACKGROUND OF THE INVENTION

[0003] Infectious pancreatic necrosis virus (IPNV) is an economicallyimportant fish pathogen. IPNV belongs to a group of viruses known asBirnaviridae (Brown (1986), Intervirology, 25:141-143). Other members ofBimaviridae include infectious bursal disease virus (IBDV) of fowl andDrosophila X virus. IPNV was discovered to be associated with a highlycontagious disease of susceptible hatchery-reared trout and salmonoids.As the name indicates, the infection among trout produces markedpancreatic necrosis, but histopathological changes sometimes also occurin adjacent adipose tissue, in renal hematopoietic tissue, in the gut,and in the liver (Wolf et al.(1960), Proc. Soc. Exp. Biol. Med.,104:105-108). Histopathological changes can also occur in renalexcretory and hematopoietic tissues, as first reported by Yasutake etal. (1965), Ann. N. Y. Acad. Sci., 126:520-530. Although renal damage isconsistent with the high titer of virus typically found in kidneys, atleast in carrier fish, focal degeneration of liver parenchymal cells inyearling Atlantic salmon which had been previously inoculated with IPNVwas also noted (Swanson and Gillespie (1979), J. Fish. Res. Board Can.,36:587-591).

[0004] IPNV shows a high degree of antigenic heterogeneity. Threedifferent stereotypes, Ab, Sp and VR299 (MacDonald and Gower (1981),Virology, 114:187-195; Okamoto et al. (1983), Eur. J. Fish Dis.,6:19-25) and ten subgroups (Heppell et al. (1992), J. Gen. Virol.,73:2863-2870) have been identified. IPNV is the etiological agent of acontagious, high mortality disease of young, hatchery-reared salmonids(Wolf et al. (1960), supra) and other non-salmonid fishes (Adair andFerguson (1981), J. Fish Dis., 4:69-76). IPNV is a doublestranded RNAvirus with four virion proteins (MacDonald and Dobos (1981), Virology,114:414-422).

[0005] Birnaviruses possess a bisegmented (A and B), double-stranded RNAgenome contained within a medium-sized, unenveloped, icosahedral capsid.Birnavirus gene expression involves the production of four unrelatedmajor genes that undergo various posttranslational cleavage to generatethree to five structural proteins (Dobos, P. 1995. Annu. Rew. Fish Dis.5, 25-54). The largest protein (90 kDa), VP1, is encoded by the smallersegment B genome, and the larger genome segment A encodes VP2 (42 kDa),VP4 (28 kDa) and VP3 (32 kDa). Genome segment A contains an additionalsmall open reading frame (ORF) which overlaps the amino terminal end ofthe polyprotein from the reading frame (Duncan, R., Nagy, E., Krell, P.J. and Dobos, P. 1987. J. Virol. 61, 3655-3664). This small ORF encodesa 17 kDa arginine-rich minor polypeptide, VP5, which is produced insmall quantities and is synthesized during the early replication cycle(Magyar, G. & Dobos, P. 1994. Virology 204, 580-589).

[0006] There are two major morphologically and biochemically distinctmodes of death in eukaryotic cells: apoptosis and necrosis (Duvall andWyllie (1986), Immunol. Today, 7:115-119). Apoptosis is a physiologicalprocess involved in normal tissue turnover that occurs duringembryogenesis, aging, and tumor regression, but pathological stimuli,such as viral infections (Gougeon and Montagnier (1993), Science,260:1269-1270), can also be triggering factors. Typically, apoptoticcell death is characterized by nuclear condensation, endonucleolyticdegradation of DNA at nucleosomal intervals (“laddering”), and plasmamembrane blebbing (Wyllie et al. (1980), Int. Rev. Cytol., 68:251-306).Necrosis is a pathological reaction that occurs in response toperturbations in the cellular environment such as complement attack,severe hypoxia, or hyperthermia. These stimuli increase the permeabilityof the plasma membrane, resulting in irreversible swelling of the cells(Wyllie et al,(1980), supra).

[0007] Apoptosis is important during embryonic development,metamorphosis, tissue renewal, hormone-induced tissue atrophy and manypathological conditions. In multi-cellular organisms, apoptosis ensuresthe elimination of superfluous cells including those that are generatedin excess, have already completed their specific functions or areharmful to the whole organism. In reproductive tissues, massive celldeath occurs under the control of hormonal signals. A growing body ofevidence suggests that the intracellular “death program” activatedduring apoptosis is similar in different cell types and conserved duringevolution. (Hengartner and Horvitz (1994), Cell, 76:1107-1114). Inaddition to being essential for normal development and maintenance,apoptosis is important in the defense against viral infection and inpreventing the emergence of cancer.

[0008] Apoptosis involves two essential steps. The “decision” step iscontrolled by the Bcl-2 family of proteins which consists of differentanti- and pro-apoptotic members. The “execution” phase of apoptosis ismediated by the activation of caspases and cysteine proteases thatinduce cell death via the proteolytic cleavage of substrates vital forcellular homeostasis.

[0009] Bcl-2 protein is a 25 kD integral membrane protein of themitochondria. Bcl-2 protein extends survival in many different celltypes by inhibiting apoptosis elicited by a variety of death-inducingstimuli (Korsmeyer (1992), Blood, 80:879-886). Overexpression of bcl-2has been related to hyperplasia, autoimmunity and resistance toapoptosis (Fang et al. (1994), J. Immunol., 153:4388-4398). Bcl-2contains a family of related genes, which includes, but is not limitedto, A1, mcl-1, bcl-w, bax, bad, bak and bcl-x. A1, mcl-1, bcl-2 andbcl-x1 (long form of bcl-x) are presently known to confer protectionagainst apoptosis and are referred to herein as “anti-apoptotic bcl-2related proteins”. In contrast, bax, bad, bak and bcl-xs (short form ofbcl-x) are presently known to promote cell death by inhibiting thisprotective effect.

[0010] Mcl-1 is one of the members of the Bcl-2 family. Like Bcl-2,Mcl-1 heterodimerizes with Bax, an accelerator of apoptosis in the Bcl-2family, and neutralizes the cytotoxicity induced by Bax in yeast (Bodruget al. (1996), Death Differ., 2:173-182). Mcl-1 is also able to protectChinese hamster ovary cells from apoptosis induced by c-mycoverexpression (Reynolds et al (1994), Cancer Res., 54:6348-6352). Thisprotein was discovered as a novel gene induced early in the induction ofdifferentiation of a human myeloid leukemia cell line (Kozopas et al.(1993), Proc. Natl. Acad. Sci. USA, 90:3516-3520). Expression of Mcl-1mRNA was rapidly up-regulated with phorbol ester in those cells followedby a rapid degradation, consistent with the presence of a mRNAdestabilization sequence in its 3′-untranslated region. The half-life ofthe Mcl-1 protein is short (Yang et al. (1995), J. Cell Biol.,128:1173-1184), which has been ascribed to the presence of two PEST(proline, glutamic acid, serine, threonine) motifs. Therefore, Mcl-I issuggested as a rapidly inducible, short-term effector of cell viability(Yang et al. (1996), J. Cell. Physiol., 166:523-536). Recently, Hong etal. (Virology, (1998), 250:76-84) reported that an El-S of IPNV Abstrain induced apoptosis in CHSE-214 cells. Hong et al.'s publication isherein incorporated by reference. In Hong et al.'s report, four kinds ofdetecting methods were used to determine whether apoptosis is involvedin fish embryonic cell death after IPNV infection: (1) assay withterminal deoxynucleotidyl transferase (TdT)-mediated end-labeling of DNAin nuclei of intact cells during virus infection; (2) assay forprocoagulant activity; (3) assay for DNA ladders; and (4) electronmicroscopic assays for the ultrastructural changes in characteristicapoptotic cells.

[0011] The results show that apoptosis precedes any detectable necroticchange in CHSE-214 cells, suggesting that apoptosis characterizes theonset of pathology in host cells and is followed by necrotic processes.Hong et al.'s report is important because previously, IPNV infection isonly viewed as caused by a necrotic process. However, Hong et al.'sreport did not provide any insights which delineate the apoptoticprocess from necrosis.

[0012] In the present invention, an investigation of apoptosis iscarried out by using CHSE214 cells infected with IPNV as a model. Theinvestigation is conducted by transfecting the cells with a pEGFP vectorwhich enables the cells to express EGFP (a variant type of GFP [greenfluorescent protein]). Based on the special characteristics of EGFPwhich can fluorescence 35 times more intense than the wild-type GFP, themorphological changes during apoptosis are monitored, which show thatIPNV causes CHSE-214-EGFP cells to undergo apoptosis, then a nontypicalapoptosis, and finally, postapoptotic necrosis in cells. The discoveryof the nontypical apoptosis stage before necrosis takes place is one ofthe novel findings in the present invention.

[0013] The present invention also provides studies of apoptosis via anMcl-1 dependent pathway. The results of the present invention indicatethat the occurrence of apoptosis is due to down regulation of the Mcl-1gene caused by viral infection. In addition, various drugs or chemicalsare tested for their capacity of preventing the down-regulation of Mcl-1protein expression by viral infection. The results show that by blockingthe down regulation of the Mcl-1 gene, the cell death caused by IPNVinfection is effectively prevented.

[0014] The present invention is important because it not only provides amodel for studies of apoptosis but also provides a means for preventingor containing widespread of IPNV infection in aquatic organisms.

SUMMARY OF THE INVENTION

[0015] The present invention provides an aquatic apoptotic cell whichcan be used as a model for studying morphological changes duringapoptosis. The aquatic apoptotic cell is induced by infecting an aquaticcell with an aquabirnavirus. The preferred aquatic cell is a fish cell.The preferred fish cell includes, but is not limited to, salmon, trout,grouper, and eel cells. The most preferred fish cell is Chinook salmonembryo cell (CHSE-214). The preferred aquabimavirus is an infectiouspancreatic necrosis virus (IPNV). The preferred IPNV is El-S of IPNV Abstrain which is isolated from Japanese eel in Taiwan (Wu et al. (1987),Bull. Inst. Zool. Acad. Sinica, 26:201-214). The infectious period ispreferred not to exceed 8 hours.

[0016] The morphological changes of the aquatic apoptotic cell duringapoptosis can be monitored by fluorescence using EGFP as a probe. EGFPis introduced into the aquatic cell by transfecting the cell with apEGFP-N1 vector. By using EGFP, a nontypical apoptotic process isdiscovered which occurs after a typical apoptosis and before thenecrosis process. This nontypical apoptosis features, including highlycondensed membrane blebbing, occurs during the middle apoptotic stage.At the pre-late apoptotic stage, membrane vesicles quickly formed,blebbed, and are finally pinched off from the cell membrane. Together,these findings show the apoptotic features at the onset of pathology inhost cells (early and middle apoptotic stages), followed secondarily bynontypical apoptosis (pre-late apoptotic stage) and then bypostapoptotic necrosis (late apoptotic stage), of a fish cell. Theresults also demonstrate that nontypical apoptosis is a component ofIPNV-induced fish cell death.

[0017] The present invention also provides agents for inducing orpreventing/rescuing apoptosis. The first agent for inducing apoptosis isIPNV itself. The second agent for inducing apoptosis is VP3, a 32-kDaprotein derived from the IPNV segment A. The nucleotide sequence of VP3is publicly available, as accession number AF291752 in NCBI gene databank; website address: www.ncbi.nlm.nih.gov. The VP3 gene can beconverted into cDNA by RT-PCT and inserted into a plasmid to transfect ahost organism or a cell line.

[0018] The first agent for preventing/rescuing apoptosis caused by IPNVor VP3 is an antisense RNA of VP3, which can be transfected into a hostor a cell line. The second agent for preventing/rescuing apoptosiscaused by IPNV or VP3 is a zfmcl-1a gene, which can be inserted into aplasmid (such as pEGFP-zfMcl-1a) for transfection into a host or a cellline.

[0019] The present invention also includes two methods for detectingapoptosis. The first method provides a means to visualize morphologicalchanges during apoptosis. The method contains the following steps: (1)transfecting the aquatic cells with a pEGFP-N1 vector; (2) infecting theaquatic cell with an aquabimavirus; and (3) monitoring the morphologicalchanges by a microscopic technique. The pEGFP-N1 vector is driven by animmediate-early promoter of human cytomegalovirus. The coding regioncontains the EGFP gene, which contains a chromophore mutation whichproduces fluorescence 35 times more intense than that of wide-type GFP(green fluorescence protein). GFP is a revolutionary molecule which canbe used to monitor gene expression and fusion protein localization invivo or in situ and in real time. GFP is from the jellyfish Aequoreavictoria. The transfected cells can be screened by G418. The preferredaquabimavirus is IPNV. The preferred aquatic cell is CHSE-214. Themicroscopic technique includes, but is not limited to, light microscopy,fluorescence microscopy, scanning electron microscopy, andimmuno-electron microscopy.

[0020] The second method provides quantitative measurements ofapoptosis. The method contains the following steps: (1) transfecting anaquatic cell with a pEGFP-N1 vector; (2) infecting the aquatic cell withan aquabimavirus; and (3) measuing EGFP in the aquatic cell and in theculture medium. EGFP is measured based upon the fluorescence intensity.The pEGFP-N1 transfected aquatic cell produces EGFP which can beevaluated by a Fluorolite 1000 (DYNEX).

[0021] Furthermore, the present invention provides methods for inducingor preventing apoptosis in vivo or in vitro. The first method forinducing apoptosis includes in vivo infection of aquatic organisms withan aquabimavirus. The preferred aquabimavirus is IPNV. The preferredIPNV is El-S of IPNV Ab strain which is isolated from Japanese eel inTaiwan (Wu et al. (1987), supra). The preferred aquatic organisms areembryos or hatchery-reared juvenile salmonids and nonsalmonid fish.Salmonid fish include, but are limited to, pacific salmon in general(Oncorhynchus sp.), such as rainbow trout (Oncorhynchus mykiss), brooktrout (Salvelinus fontinolis), chinook salmon (Oncorhynchustshawytscha), coho salmon (Oncorhynchus kisutch), sockeye salmon(Oncorhynchus nerca) and Atlantic salmon (Salmo salar). Nonsalmonid fishincludes carp, perch, pike, eels and char. Certain mollusks andcrustaceans can also be infected with IPNV.

[0022] This method also applies to in vitro transfection of IPNV intofish cell lines, such as CHSE-214 (an embryo cell line from salmon). Thepreferred temperature for infected cultures is at 18° C. The inductionof apoptosis by aquabimavirus can be prevented by pretreatment ofcyclohexamide (protein synthesis inhibitor), genistein (tyrosine kinaseinhibitor), and EDTA (cation chelator) prior to viral infection.

[0023] The second method for inducing apoptosis includes transfection ofa VP3 gene-containing plasmid into a host, which may be anaquatic/vetebrate organism or cell line, to overexpress VP3 in the host.VP3 is a 32-kDa protein derived from the IPNV segment. The nucleotidesequence of VP3 is publicly available, as accession number AF 291752 inNCBI gene data bank; website address: www.ncbi.nlm.nih.gov.

[0024] The VP3 gene of IPNV can be converted into DNA by RT-PCR andconstructed into a plasmid containing EGFP to form a pEGFP-VP3 bytechniques which are known to persons with ordinary skill in the art.The preferred aquatic host includes salmonids and nonsalmonid fish.Salmonid fish include, but are limited to, pacific salmon in general(Oncorhynchus sp.), such as rainbow trout (Oncorhynchus mykiss), brooktrout (Salvelinus fontinolis), chinook salmon (Oncorhynchustshawytscha), coho salmon (Oncorhynchus kisutch), sockeye salmon(Oncorhynchus nerca) and Atlantic salmon (Salmo salar). Nonsalmonid fishincludes carp, perch, pike, eels, zebrafish and char. The preferredvetebrate host includes rat, hamster and human. The preferred aquatic orvertebrate organism is embryo. The pEGFP-VP3 can transfect with liposometo cell lines or a vertebrate embryo by microinjection by commonly knownmethods. The preferred cell lines for induction of apoptosis by VP3include CHSE214 (an embryo cell line from salmon), NIH3T3 (micefibroblast), CHO (Chinese hamster ovary cell), and Hepa 3b and Hepa G2cells (human liver tumor cells).

[0025] The third method for inducing apoptosis include down regulatingMcl-1 gene expression. The down regulation of Mcl-1 gene expressioncorrelates to IPNV replication. The preferred aquatic organisms arefish, in particular fish cells derived from salmon, trout, grouper, andeel. The down regulation of Mcl-1 gene expression can be blocked bydrugs such as cyclohexamide (protein synthesis inhibitor), genistein(tyrosine kinase inhibitor), and EDTA (cation chelator). These drugshelp to maintain Mcl-1 expression level and block the induction of DNAinternucleosomal cleavage (i.e., blocking the intense DNA ladderingpattern), so as to rescue or delay the apoptotic cell death process.

[0026] The first method for preventing or rescuing apoptosis by IPNVinfection or VP3 transfection requires transfecting an antisense RNA ofVP3 into a host, which may be an aquatic/vertebrate organism or cellline. The preferred aquatic host includes salmonids and nonsalmonidfish. Salmonid fish include, but are limited to, pacific salmon ingeneral (Oncorhynchus sp.), such as rainbow trout (Oncorhynchus mykiss),brook trout (Salvelinus fontinolis), chinook salmon (Oncorhynchustshawytscha), coho salmon (Oncorhynchus kisutch), sockeye salmon(Oncorhynchus nerca) and Atlantic salmon (Salmo salar). Nonsalmonid fishincludes carp, perch, pike, eels, zebrafish and char. The preferredvetebrate host includes rat, hamster and human. The preferred aquatic orvertebrate organism is embryo. The pEGFP-VP3 can transfect an aquatic ora vertebrate embryo by microinjection by commonly known methods. Thepreferred cell lines include CHSE-214 (an embryo cell line from salmon),NIH3T3 (rat fibroblast), CHO (hamster embryoic cells), and Hepa 3b andHepa G2 cells (human liver tumor cells).

[0027] The second method for preventing or rescuing apoptosis from IPNVinfection requires transfecting a zfMcl-1a into a host, which may be anaquatic/vertebrate organism or cell line. The nucleotide sequence ofzfMcl-1a is publicly available, which is ZfMcl-1a accession number AF231016 as published in NCBI gene data bank; website address:www.ncbi.nlm.nih.gov. The zfMcl-1a can be transfected into the host bymicroinjecting a zfMcl-1a-containing plasmid such as pEGFP-zfMcl-1a orpEGFP-C1. The preferred aquatic host includes salmonids and nonsahnonidfish. Salmonid fish include, but are limited to, pacific salmon ingeneral (Oncorhynchus sp.), such as rainbow trout (Oncorhynchus mykiss),brook trout (Salvelinus fontinolis), chinook salmon (Oncorhynchustshawytscha), coho salmon (Oncorhynchus kisutch), sockeye salmon(Oncorhynchus nerca) and Atlantic salmon (Salmo salar). Nonsalmonid fishincludes carp, perch, pike, eels, zebrafish and char. The preferredvertebrate host includes rat, hamster and human. The preferred aquaticor vertebrate organism is embryo. The pEGFP-VP3 can transfected anaquatic or a vertebrate embryo by microinjection by commonly knownmethods. The preferred cell lines for preventing or rescuing apoptosisinclude CHSE-214 (an embryo cell line from salmon), NIH3T3 (micefibroblast), CHO (Chinese hamster ovary cell), and Hepa 3b and Hepa G2cells (human liver tumor cells).

BRIEF DESCRIPTION OF THE FIGURES

[0028]FIG. 1 shows the dynamics of the sequential morphological changesvisualized by EGFP in living cells infected with IPNV. Monolayercultures of CHSE-214 cells were transfected with pEGFP-N1 usingLipofectin and selected with G418. Cells were infected with virus (MOIof 1), and virus-infected cells were sequentially observed byfluorescence microscopy from 0 to 7 h post infection (p.i.) Scale bar, 3μm. The arrows indicate the formation of membrane vesicles (MV) from theapoptotic cell.

[0029]FIG. 2 shows the analysis of DNA fragments in CHSE-214-EGFP cellsafter being infected with IPNV EI-S (MOI of 1). DNA was isolated fromuninfected CHSE-214 cells as a negative control after 0 h (lane 2) and 8h (lane 3) of incubation and from cells infected for 8 h with an MOI of1 of El-S (lane 4), electrophoresed through 1.2% agarose gels, andvisualized by ethidium bromide staining. Lane 1 contained molecular sizemarkers (1-kb DNA ladder).

[0030]FIG. 3 shows the scanning electron micrographs of CHSE-214 cells.(A) Negative control CHSE-214 cell. (B) Pre-late apoptotic CHSE-214cell. The formation of membrane vesicles (MV) from the apoptotic cell isindicated by arrows. (C) Middle-late apoptotic cell. The formation ofsmall holes is indicated by arrows. (D) Late apoptotic cell. Small holesleft on the surfaces of apoptotic bodies from the IPNV-treated group areindicated by arrows. Scale bar, 1.5 μm.

[0031]FIG. 4 shows the patterns of EGFP release by CHSE-214-EGFP cellsinfected with IPNV. (A) shows the EGFP release patterns after IPNVinfection by Western blotting. CHSE-214-EGFP cells were infected withIPNV (MOI of 1). Samples were electrophoresed on an SDS-polyacrylamidegel and electroblotted to a nitrocellulose membrane. The membrane eithercontained a rabbit polyclonal antiserum directed against EGFP (part aand c) or was stripped and reprobed with a mouse IgG monoclonal antibodydirected against actin (part b). The chemiluminescent signal was imagedon Kodak XAR-5 film using a 3-min (part a), 1.5-min (part b), or 30-min(part c) exposure. (a) Lanes: 1:0.45 μg of wild-type GFP; 2 to 7:30 μgof virus-infected CHSE-214 cell lysate at 0, 2, 4, 6, 8, and 16 hpostinfection (p.i.), respectively. (b) The nitrocellulose membrane inpart a was stripped and reprobed with anti-actin monoclonal IgG. (c) A30-μg sample of supernatant protein of IPNV-infected CHSE-214-EGFP cellsat 0, 2, 4, 6, 8, 10, 12, and 24 h p.i., respectively. (B) shows therate of EGFP release by CHSE-214-EGFP cells infected with IPNV. Cellularand culture medium EGFP samples were prepared for assay in EGFP releaseexperiments. About 10⁵ cells per ml were seeded on a 60-mm petri dishand incubated for more than 20 h. Cells that received virus at an MOI of1 were incubated for 0, 2, 4, 6, 8, 10, 12, and 24 h p.i. At the end ofeach incubation time, the IPNV-infected CHSE-214 cells and culturemedium were collected to determine the concentration of retained EGFP.Both 5 μg of lysed virus-infected cells per sample and 30 μg ofsupernatant medium per sample were counted by a Fluorolite 1000. TheEGFP concentrations of the lysed cells and supernatant were evaluated byusing a Fluorolite 1000 to compare them with standard GFP protein anddividing by 35.

[0032]FIG. 5 shows the immunoelectron micrographs of ultrathin sectionsof CHSE-214EGFP cells that were uninfected or infected with IPNV andlabeled with anti-GFP IgG. (A) Normal CHSE-214-EGFP cell used as anegative (N) control on which labeled EGFP is present (arrows) and EGFPformed dimers. (B) CHSE-214-EGFP cell infected with IPNV (MOI of 1) at 8h p.i. upon which labeled EGFP is present (small arrows). Nontypicalapoptotic morphological changes were observed at this pre-late apoptoticcell stage such as the formation of membrane vesicles (MV) (large, longarrow) and, finally, the MV pinching off from the plasma membrane of theapoptotic cell (large, short arrow).

[0033]FIG. 6 is a western blot assay which shows the effect of chemicalinhibitors on EGFP release. Protein synthesis inhibitors, serineproteinase inhibitors, tyrosine kinase inhibitors, and a cation chelatorwere added to CHSE-214-EGFP cells before infection with IPNV (MOI of 1).After infection, the cells were incubated for 16 h. Samples wereelectrophoresed on a 12% SDS-polyacrylamide gel and electroblotted to anitrocellulose membrane. Antigen-specific signals were detected witheither rabbit anti-GFP serum (A and C) or a mouse IgG monoclonalantibody directed against actin (B). The chemiluminescent signal wasimaged on Kodak XAR-5 film by using a 5-min (A), a 1-min (B), or a30-min (C) exposure. (A) Lanes: 1, 0.2 μg of recombinant wild-type GFPas a positive control; 2, normal CHSE-214-EGFP cell lysate; 3, 30 μg ofcell lysate protein corresponding to IPNV infection; 4-9, 30 μg of celllysate protein corresponding to pre-treatment with cycloheximide (CHX)(100 μg/ml), aprotinin (400 μg/ml), leupeptin (400 μg/ml), genistein(100 μg/ml), tyrphostin (100 μg/ml), and EDTA (2 mM) and then followedby virus infection for 16 h, respectively. (B) The nitrocellulosemembrane from panel A was stripped and reprobed with an actin antibody.(C) Lanes: 1, 100 ng of wide-type GFP; 2, 30 μg of supernatant mediumprotein from normal CHSE-214 cells; 3, 30 μg of supernatant mediumprotein from IPNV-infected cells at 16 h p.i. 4-9, 30 μg of supernatantmedium protein corresponding to treatment with cycloheximide (CHX) (100μg/ml), aprotinin (400 μg/ml), leupeptin (400 μg/ml), genistein (100μg/ml), tyrphostin (100 μg/ml), and EDTA (2 mM) and then followed byvirus infection for 16 h, respectively.

[0034]FIG. 7 illustrates the morphological changes induced in fish cellsby IPNV infection. (A) Normal attached cell. In the early stage ofapoptosis, the cell detaches from the extracellular martix (A to B, 0 to3 h p.i.). In the middle stage, the apoptotic cell is rounded up (A toB, 3 to 6 h p.i.). To enter this pre-late apoptotic stage (B to C, 6 to7 h p.i.), there is a rapid process which follows that includes MVformation and MV pinching off from the plasma membrane. In the middlestage, the apoptotic cell is left with small holes in the cell membrane(C to D, 7 to 8 h p.i.). Finally, in the late apoptotic stage, eithermembrane-bound apoptotic bodies (D to E, 8 to 12 h p.i.) are formed or apostapoptotic necrosis process occurs (D to F, 8 to 12 h p.i.) in whichthe condensed chromatin encloses the nuclear membrane.

[0035]FIG. 8 shows the scanning electron micrographs of CHSE-214 cells.(A) Uninfected CHSE-214 cells. (B) IPNV-infected CHSE-214 cells, showingmembrane blebbing in the cell which is a typical indication ofapoptosis.

[0036]FIG. 9 is an analysis of DNA fragments of IPNV El-S-infectedCHSE-214 cells by 1.2% agarose gel electrophoresis. Lane 1 contains themolecular weight markers used in the gel (1-kb DNA ladder from MBIFermentas Inc. USA, for sizing liner fragments ranging in size from 500bp to 1 kb). Lane 2 is a negative control using DNA isolated fromuninfected CHSE-214 cells for 0 hour incubation. Lanes 3-5 represent DNAisolated from CHSE-214 cells after being infected by IPNV for 4, 8 and12 hours with a MOI (multiplicity of infection) of 1. The agarose gelwas stained with ethidium bromide.

[0037]FIG. 10 shows the detection of major proteins of IPNV El-S strainon Western blots. Samples were electrophoresed on a SDS-polyacrylamidegel and electroblotted to a nitrocellulose membrane. Antigen-specificsignals were detected with a rabbit anti-El-S virion antiserum and agoat anti-rabbit IgG conjugated to alkaline phosphatase. Thechemiluminescent signal was imaged on Kodak X-OMAT film (Eastman Kodak)with a 1.5-min exposure. Lanes 1-7 correspond to a MOI of 1 infectedcells for 0, 2, 4, 6, 8, 10 and 24 h post-infection.

[0038]FIG. 11 shows the detection of Mcl-l protein in CHSE-214 by IPNVinfection on Western blots. CHSE-214 cells were infected with IPNV (MOIof 1). Samples were electrophoresed on a SDS-polyacryamide gel andelectroblotted to a nitrocellulose membrane. The nitrocellulose membranewas stained with either a rabbit polyclonal antiserum directed againsthuman MCL-1 (Pharmingen) or mouse monoclonal IgG antibodies directedagainst actin (Amershan) (B). The chemiluminescent signal was imaged onKodak XAR-5 film using a 3-min (A) and 1.5-min (B) exposure. (A) Lanes1-7, 30 μg of virus infected CHSE-214 cell lysate corresponding to 0, 2,4, 6, 8, and 24 h postinfection, respectively. (B) The nitrocellulosemembrane of (A) was stripped in stripping buffer containing 62.5 mMTris-HCl (pH 6.8), 3.0% (w/v) SDS, and 50 mM 1,4-dithiothreitol for 30min at 55° C. with gentle shaking to remove the primary (Mcl-1) andsecondary antibodies (peroxidase-labeled goat anti-rabbit conjugate).The blots were then washed four times for 10 min each time in PBScontaining 0.1% (v/v) Tween 20 and reprobed with mouse actin monoclonalantibody (1/1500, Chemicon) and a 1:7500 dilution of aperoxidase-labeled goat anti-mouse conjugate (Amersham).

[0039]FIG. 12 shows the effect of blocking viral protein expression bydrugs on Western blots. After adding protein synthesis inhibitor, serineproteinase inhibitors, tyrosine kinase inhibitors and a cation chelator,CHSE-214-EGFP cells were infected with IPNV (MOI of 1) then incubatedfor 16 h at 18° C. Samples were electrophoresed on a 12%SDS-polyacrylamide gel and electroblotted to a nitrocellulose membrane.Antigen-specific signals were detected with a rabbit anti-EI-S virionantiserum (A) or with a rabbit anti-human Mcl-1 antiserum (Pharmingen)(B) and with a mouse monoclonal IgG antibody directed against mouseactin (Chemicon) (C). (A) Lane 1, normal CHSE-214 cell lysate; lane 2,IPNV infected cell lysate at 16 h postinfection; lanes 3-8, 30 μg ofcell lysate, to which was added cycloheximide (100 μg/ml), aprotinin(400 μg/ml), leupeptin (400 μg/ml), genistein (100 μg/ml), tyrphostin(100 μg/ml) and EDTA (2 mM), followed by IPNV infection and incubatedcells for 16 h, respectively. (B,C) The nitrocellulose membrane from (A)was stripped and reprobed with Mcl-1 or an actin antibody, respectively.

[0040]FIG. 13 shows the effect of certain drugs on preventing host cellDNA internucleosomal cleavage prior to IPNV El-S (MOI of 1) infection.Lane 2: DNA isolated from uninfected CHSE-214 cells as a negativecontrol. Lane 3: cells infected for 16 h with an MOI of 1 of El-S,respectively, at 16 h postinfection. Lanes 6 and 7: CHSE-214 cellstreated with aprotinin (400 μg/ml) in the absence and presence ofinfection by an MOI of 1 of El-S, respectively, at 16 h postinfection.Lanes 8 and 9: CHSE-214 cells treated with leupeptin (400 μg/ml) in theabsence and presence of infection by an MOI of 1 of El-S, respectively,at 16 h postinfection. Lanes 10 and 11: CHSE-214 cells treated withgenistein (100 μg/ml) in the absence and presence of infection by an MOIof 1 of El-S, respectively, at 16 h postinfection. Lanes 12 and 13:CHSE-214 cells treated with tyrphostin (100 μg/ml) in the absence andpresence of infection by an MOI of 1 of El-S, respectively, at 16 hpostinfection. Lanes 14 and 15: CHSE-214 cells treated with 2 mM EDTA inthe absence and presence of infection by an MOI of 1 of El-S,respectively, at 16 h postinfection. All samples were electrophoresedthrough 1.2% agarose gels and visualized by ethidium bromide staining.Lane 1 contains the molecular weight marekers used in the gel (1 kb DNAladder from MBI for sizing linear fragments ranging in size from 500 bpto 1 kb).

[0041]FIG. 14 shows that VP 3 antisense RNA could prevent cell deathduring IPNV infection in CHSE-214 cell lines at 24 hours post-infection(p.i.). pA200, pA400, and pA700 are plasmids containing various length(nucleotides) of VP3 antisense RNA (see infra for details). psDNA3 is acommercially available plasmid from Invitrogen, U.S.A., which is used asnegative control.

[0042]FIG. 15 shows that VP3 anti-sense RNA could knock-down VP3 deathfunction and enhances the host cell survival during IPNV infection. Thesurvival rate (%) of CHSE-214 cells with or without VP3 antisense RNAenhances cell survival during a 24 hours post infection with IPNV-E1 S.pcDNA3 (24%); pA200 (67%); pA400 (42%); pA700 (48%).

[0043]FIG. 16 shows the induction of apoptosis in NIH3T3 cells due tooverexpression of IPNV VP3. A fused gene, EGFP-VP3 has been generated inplasmid. The EGFP-VP3 plasmid was then transfected to NIH3T3 cells inthe together with lipofectamine. The micrograph shown in the figure wastaken 24 hours after IPNV infection that apoptotic cell indicated bylong arrow and apoptotic body indicated by short arrow.

[0044]FIG. 17 shows % of apoptotic positive cells in NIH3T3 cell line 24or 36 hours after being transfected with EGFP-C1 or EGFP-VP3:transfected with EGFP-C 1 (all 2% at 24 or 36 h); transfected withEGFP-VP3 (13% at 24 h and 20.5% at 36 h).

[0045]FIG. 18 shows induction of intemucleosomal cleavage by VP3 inzebrafish embryonic cell. DNA fragments was examined in 1.2% agarose gelafter microinjection of pEGFP-VP3 or pEGFP-C1 into zebrafish embryos. L:Live zebrafish embryo; D: Dead zebrafish embryo. Lane 1: DNA sizemarker; Lane 2: Live embryo after receiving pEGFP-C1 for 12 hours(control); Lane 3: Dead embryo after receiving pEGFP-C1 for 12 hours(control); Lane 4: Live embryo after receiving pEGFP-VP3 for 12 hours;Lane 5: Dead embryo after receiving pEGFP-VP3 for 4 hours; Lane 6: Deadembryo after receiving pEGFP-VP3 for 12 hours (which shows intense DNAfragmentation).

[0046]FIG. 19 shows VP3-induced apoptotic cell death in zebrafishembryos and damage in the somite after microinjection with pEGFP-VP3 for24 hours.

[0047]FIGS. 19A and B are zebrafish embryos after injected with pEGFP-C1(negative controls), in which FIG. 19A was brightfield image (takenunder light microscope) and FIG. 19B was fluorescence image (taken underfluorescence microscope);

[0048]FIGS. 19C and D are injected with pEGFP-VP3. Shown in FIG. 19D areareas containing flattened dead cells (

) and damaged somite (

).

[0049]FIG. 20 shows microinj ection of pEGFP-C1 (A and B) and pEGFP-VP3(C and D) into zebrafish embryo for 48 hours. FIGS. 20A and C werefluorescence image (taken under fluorescence microscope);

[0050]FIGS. 20B and D were brightfield image (taken under lightmicroscope). Severe damages in somite (as shown by arrows) were seen inembryos after microinjected with pEGFP-VP3 for 48 hours.

[0051]FIG. 21 shows the induction of embryonic death by VP3 and therescue of embryonic death by VP3 antisense RNA. Survival rate isdifferences in % of number of alive zebrafish embryos aftermicroinjection of pEGFP-C1, pEGFP-VP3, or pEGFP-VP3 plus antisense RNAof VP3 to the embryos. □ Normal control (88%); pEGFP-C1 (68%); pEGFP-VP3(42%); pEGFP-VP3+antisense RNA of VP3 (82%, N all more than 100 andrepeat three times).

[0052]FIG. 22 shows VP3-induced zebrafish embryonic defect and theprevention of VP3induced zebrafish embroyonic defect by VP3 antisenseRNA. The bar graph shows the % of defected embryo after microinjectionof pEGFP-C1, pEGFP-VP3, or pEGFP-VP3 plus antisense RNA of VP3, to theembryos for 60 hours. Normal control (1%); pEGFP-C1(4%);pEGFP-VP3(12.5%); pEGFP-VP3+antisense RNA of VP3 (29%).

[0053]FIG. 23 shows VP3 cell death function blocked by antisense RNA. Acomposite of two fluorescence micrographs of zebrafish embryos shown in(D) that was microinjected with pEGFP-VP3 for 12 hours. Arrow shown in(D) indicates materials leaked out of the dead cell. The embryo shown in(F) was microinjected with pEGFP-VP3 and antisense RNA for 12 hours.

[0054]FIG. 24 shows the anti-apoptosis effect of zfMcl-1 a (to preventembryonic defect) during VP3 expression. The bar graph shows the % ofdefected embryo after microinjection with normal control, pEGFP-C1,pEGFP-VP3, pEGFP-VP3 plus pEGFP-Mcl-1a; or pEGFPVP3 plus pEGFP-Bcl-xLfor 36 hours. ¦ Normal control (1%); pEGFP-C1 (5%); pEGFPVP3 (30%);pEGFP-VP3 +pEGFP-Mcl-1a (7.5%); pEGFP-VP3 +pEGFP-Bcl-xL (26%). BothMcl-1a and Bcl-xL were derived from zebrafish.

[0055]FIG. 25 shows the anti-apoptotic effect of the Bcl-2 familyzfMcl-1a on prevention of embryonic death induced by overexpression ofVP3. The bar graph shows the % of alive embryos after microinjectionwith normal control, pEGFP-C1, pEGFP-VP3, pEGFP-VP3 plus pEGFP-Mcl-1a;or pEGFP-VP3 plus pEGFP-Bcl-xL for 36 hours. Normal control (91%);pEGFP-C1 (75%); pEGFP-VP3 (37.5%); pEGFP-VP3 +pEGFP-Mcl-1a (76%);pEGFP-VP3 +pEGFP-Bcl-xL (39%). Both Mcl-1a and Bcl-xL were derived fromzebrafish.

DETAILED DESCRIPTION OF THE INVENTION

[0056] Apoptosis is a morphologically distinct cell death thatspontaneously occurs in many different tissues under various conditions(Falcieri et al. (1994), Scan. Microsc. 8, 653-666). It occurs indistinctly separated cells and progresses very rapidly, never causingexudative inflammation in tissues. Typically, apoptosis is characterizedmorphologically by cell shrinkage and hyperchromatic nuclear fragmentsand biochemically by chromatin cleavage into nucleosomal oligomers.

[0057] Infectious pancreatic necrosis virus (IPNV) is the prototypevirus of the family Birnaviridae. The virus is capable of infecting anumber of different hosts and has a worldwide presence. IPNV isespecially susceptible to salmonids, particularly trout, salmon, carp,perch, pike, and eel. The most susceptible fish for IPNV are trout andsalmon, such as rainbow trout (Oncorhynchus mykiss), brook trout(Salvelinus fontinolis), chinook salmon (Oncorhynchus tshawytscha), cohosalmon (Oncorhynchus kisutch), sockeye salmon (Oncorhynchus nerca) andAtlantic salmon (Salmo salar).

[0058] IPNV contains a bisegmented double stranded RNA genome. Thelarger genome segment A (approximately 3100 bp) of IPNV has two openreading frames: (1) one large

[0059] IPNV contains a bisegmented double stranded RNA genome. Thelarger genome segment A (approximately 3100 bp) of IPNV has two openreading frames: (1) one large 2916 bp ORF encoding a 106 kD polypeptidewhich can be cleaved into at least three protein; and (2) oneoverlapping ORF of 444 bp encoding a 17kD arginine rich polypeptide.

[0060] The three proteins encoded by the large ORF include: (1) a 60K to62K precursor (pVP2) of the 52K to 54K major capsid protein VP2; (2) a29K non-structural protein (NS); and (3) a 31K minor capsid protein VP3.VP3 is believed to be located internally, associated with the RNA, butmay be partly exposed on the surface of the capsid. The localization ofthe 17 kD polypeptide is not known.

[0061] The smaller B segment (approximately 2900 bp) encodes a singegene product (VP1) with a molecular weight of approximately 94K,presumed to be the viral RNA polymerase. VP1 is present as freepolypeptide in the virion and as genome-linked protein, VPg.

[0062] IPNV is a contagious disease, which has particular importance ofits effects on hatchery raised fish. The virus replicates in thecytoplasm and a single cycle of replication normally takes 16-20 h at22° C., which results in a characteristic cytopathic effect (CPE).

[0063] Recently, Hong et al. (1998), supra, reported that in Chinooksalmon embryo (CHSE214) cells after being infected by IPNV, apoptosisoccurred prior to necrosis. Hong et al. were able to demonstrate thatduring apoptosis, no cell hydration took place, but nuclear andcytoplasmic condensation appeared, which was followed by the formationof numerous membrane-bound cell fragments termed “apoptotic bodies.” Inaddition, in contrast to necrosis, the nuclear organization duringapoptosis was completely lost, and profound chromatin rearrangementstook place, followed by the formation of a variable number of compact,electron-dense micronuclei. However, despite the extensive nuclearchanges, both and undergoes a “secondary” necrosis.

[0064] In the present invention, further investigation into theapoptotic processes has been carried out. The investigation can besubdivided in two embodiments: (1) Monitoring morphological changesduring apoptosis; and (2) Control of cell death via an Mcl-1 dependentpathway.

[0065] Embodiment 1: Monitoring Morphological Changes During Apoptosis

[0066] Green fluorescent protein (GFP) from the jellyfish AequoreaVictoria is a revolutionary report molecule for monitoring geneexpression and fusion protein localization in vivo or in situ and inreal time. One of the most useful aspects of GFP for biological studiesis that it can be monitored in living cells. In the present study, avariant type of GFP (EGFP) serves as a marker for visualizing thedynamic apoptotic cell morphological changes and for tracing membraneintegrity changes during apoptosis by fluorescence microscopy, as wellas for quantitation of the intra- and extracellular release of EGFPduring apoptosis by western blotting and fluorometry.

[0067] The use of EGFP to study the apoptotic process is illustrated inthe following experimental designs, results, and discussion:

[0068] (A) Experimental Designs

[0069] (1) Wild-Type CHSE-214 Cells, CHSE-214-EGFP Cells, and Viruses

[0070] Chinook salmon embryo cells (CHSE-214) were obtained fromAmerican Type Culture Collection (ATCC). Cells were grown at 18° C. asmonolayers in plastic tissue culture flasks (Nunc) using Eagle's minimumessential medium (MEM) supplemented with 10% (v/v) fetal calf serum and25 μg/ml gentamicin. GFP-producing cells were obtained by transfectionof CHSE-214 cells with a pEGFP-N1 vector and selection with G418. Inthese vectors, transcription of insert sequences is driven by theimmediate-early promoter of human cytomegalovirus. The coding regioncontains the EGFP gene, which contains a chromophore mutation whichproduces fluorescence 35 times more intense than that of wild-type GFP.

[0071] The virus isolated, El-S, a member of the Ab strain of IPNV, wasobtained from Japanese eel in Taiwan. El-S was propagated in CHSE-214cell monolayers at a multiplicity of infection (MOI) of 0.01 particlesper cell. Infected cultures were incubated at 18° C. until an extensivecytopathic effect was observed. The cells were scraped into a tube withthe tissue culture medium and chilled on ice, and the cells were thensonicated. This virus stock (5×10⁷ to 1×10⁸ PFU/ml) was dispensed intol-ml samples and stored at −70° C. Virus plaque assays were performed onconfluent monolayers of CHSE-214 cells that were infected with the virussolution for 1 h at room temperature, overlaid with 0.6% agarosecontaining 2.5 μg of trypsin per ml, and incubated for 3 days at 18° C.cells were then stained with 1% crystal violet in 20% ethanol (8).

[0072] (2) Immunoblotting

[0073] About 10⁵ cells per ml were seeded on a 60-mm-diameter petri dishand allowed to grow for more than 20 h. The cell monolayers were rinsedtwice with phosphate-buffered saline (PBS), after which they wereinfected at an MOI of 1 and incubated for 0, 2, 4, 6, 8, 10, 12, and 24h post infection (p.i.). Uninfected control cells were also incubatedfor the same periods of time. At the end of each incubation time, theculture medium was aspirated. The cells were washed with PBS and thenlysed in 0.3 ml of lysis buffer (10 mM Tris base, 20% glycerol, 10 mMsodium dodecyl sulfate [SDS], 2% β-mercaptoethanol, pH 6.8).

[0074] Proteins were separated by SDS-polyacrylamide gelelectrophoresis, electroblotted, and subjected to immunodetection asdescribed by Kain et al. (1994), BioTechniques, 17:982-987. Blots wereincubated with a 1:7,500 dilution of an immunoglobulin fraction(Clontech) and a 1:1,500 dilution of a peroxidase-labeled goatanti-rabbit conjugate (Amersham). Chemilumine-scence detection wasperformed in accordance with the instructions provided with the WesternExposure Chemiluminescent Detection System (Amersham). Chemiluminescentsignals were imaged by exposure of Kodak XAR-5 film (Eastman Kodak,Rochester, N.Y.). Stripping and reprobing of the Western blot andremoval of the primary and secondary antibodies from blot were achievedby incubation in stripping buffer containing 62.5 mM Tris-HCl (pH 6.8),3.0% (wt/vol) SDS, and 50 mM 1,4dithiothreitol for 30 min at 55° C. withgentle shaking. The blot was washed three times in PBS containing 0.1%(vol/vol) Tween 20 for 10 min each time and reprobed with antibodiesbeginning at the membrane blocking step.

[0075] Experiments examining the potency of drugs for preventingmorphological change and blocking membrane integrity loss and thoseexamining subsequent EGFP retention during virus infection andincubation were carried out as described above, except that extra CHX(10 μg/ml), aprotinin (400 μg/ml), leupeptin (400 μg/ml), genistein (100μg/ml), tyrphostin (100 μg/ml), and EDTA (2 mM) were added to CHSE-214cells before virus infection and incubation for 16 h. At the end of theincubation period, cells were harvested and samples were analyzed byWestern blotting.

[0076] (3) Fluorescence Microscopy

[0077] A CHSE-214-EGFP monolayer infected with IPNV (MOI=1) was examinedby light and fluorescence microscopy using an Olympus IX70 microscopeequipped with a BP450480 pass excitation filter and a BA515 barrieremission filter for observation of EGFP fluorescence. Photographs weretaken with a C-35 AD-4 camera using Kodak Ektachrome 200 film.

[0078] (4) DNA Preparation and Gel Electrophoresis

[0079] About 10⁵ cells per ml were seeded on a 60-mm-diameter petri dishand allowed to grow for more than 20 h. The cell monolayers receivedvirus at an MOI of 1.0 and were incubated for 8 h. Uninfected controlcells were also incubated for 8 h. The two groups were used for DNAfragmentation studies. At the end of incubation, the cells were lysedwith lysis buffer (10 mM Tris-HCl, 0.25% Triton X-100, 1 mM EDTA, pH7.4). After treatment with phenol-chloroform-isoamyl alcohol (25:24:1),the DNA was precipitated in the presence of 0.3 M sodium acetate andcold absolute ethanol at -70° C. for 2 h and then resuspended in 10 mMTris-HCl (pH 7.4)-1 mM EDTA. Aliquots of 20 μl containing approximately5 to 10 μg of DNA were then electrophoresed in 1.2% agarose gels for 2 hat 40 V. Gels were stained with ethidium bromide and photographed underUV transillumination.

[0080] (5) Scanning Electron Microscopy

[0081] Scanning electron microscopy analysis was carried out by cellseeding on a twochamber slide. CHSE-214 cells were infected with virusat an MOI of 1 and incubated for 0, 4, 8, and 12 h. At the end point,cells were washed twice with PBS and fixed with 2.5% glutaraldehyde in0.1 M phosphate buffer. Samples were postfixed with OSO₄, dehydrated inethanol, critical point dried, and gold sputtered. A Philips 515scanning electron microscope was used to examine the specimens.

[0082] (6) Immunoelectron Microscopy

[0083] CHSE-214-EGFP cells were infected at an MOI of 1. Infected anduninfected control cells were harvested 8 h after infection.Thin-section electron microscopy and immunogold labeling were carriedout as described by McNulty et al. (1990), Avian Pathol., 19:67-73. Thegrids were stained with a 1:1,000 dilution of GFP-specific polyclonalantiserum and a 1:50 dilution of a 15-nm gold-labeled goat anti-rabbitimmunoglobulin G conjugate.

[0084] (7) Quantitation of EGFP Release by CHSE-214-EGFP Cells

[0085] Cellular EGFP and culture medium EGFP protein samples wereprepared for assay in EGFP release experiments. About 10⁵ cells per mlwere seeded on a 60-mm petri dish for more than 20 h. Cell monolayerswere rinsed twice with PBS and then cultured in 3 ml of 10%FCS-containing MEM. Uninfected cells used as a normal control and cellsthat received virus at an MOI of 1 were incubated for 0. 2, 4, 6, 8, 10,12, and 24 h p.i. At the end of each incubation period, the culturemedium was collected to determine the concentration of retained EGFP.Cells were washed with PBS and then lysed in 0.3 ml of lysis buffer (10mM Tris base, 20% glycerol, 10 mM SDS, 2% β-mercaptoethanot, pH 6.8).

[0086] The assay procedure was as follows. First, recombinant GFPpurchased from Clontech was used as the standard. The GFP standard wasdiluted from 1 μg/0.1 ml to 0.001 μg/0.1 ml with 10% FCS-containing MEM.Second, 5 μg of lysed cells per sample was diluted with 10%FCS-containing MEM to a final volume of 100 μl. Third, the supernatantwas assayed, and 30 μg of supernatant per sample was diluted with 10%FCS-containing MEM to a final volume of 100 μl. Protein concentrationwas determined by the dye-binding method of Bradford using acommercially available kit (Bio-Rad, Richmond, Calif.) with bovine serumas the standard. Fourth, the fluorescence intensity of three groupsamples was counted by a Fluorolite 1000 (DYNEX). The EGFPconcentrations of the lysed cells and supernatant were evaluated bycomparing them with that of the GFP standard by using a Fluorolite 1000and dividing by 35.

[0087] (B) Results

[0088] (1) Visualization of Dynamic Morphological Changes by EGFP

[0089]FIG. 1 shows the sequential morphological changes that occurred inCHSE-214-EGFP cells during virus infection (MOI=1). These events weredivided into three stages. First (early stage), detachment of theCHSE-214-EGFP cell matrix was initiated between 0 and 3 h p.i. Second(middle stage), the whole cell was rounded up and appearedmorphologically more compact. In this period (3 to 6 h p.i.), the cellvolume decreased to one-third of its original size and the fluorescenceintensity was enhanced. In the third (pre-late) stage, the cells at 6 to7 h P.i. quickly underwent severe morphological changes. Membranevesicles (MV) were formed from the plasma membrane, and these vesicleseventually blebbed and finally pinched off from the cell membrane.

[0090] (2) Induction of Internucleosomal Cleavage by IPNV inCHSE-214-EGFP Cell

[0091] The effect of IPNV infection on host DNA in CHSE-214-EGFP cellswas examined by agarose gel electrophoresis. Virus (MOI=1) infectedcells were examined for evidence of intemucleosomal fragmentation. DNAfragmentation is a well-defined biochemical marker of apoptosis. FIG. 2shows the results of agarose gel electrophoresis which demonstrates thatintense internucleosomal fragmentation of DNA, a pattern highly specificto apoptosis, occurred in CHSE-214-EGFP cells infected with IPNV. TheIPNV induced DNA fragmentation at 8 h p.i. is shown in FIG. 2, lane 4.The negative control which showed no DNA fragmentation at 0 and 8 h ofincubation is shown in FIG. 2, lanes 2 and 3. Lane 1 of FIG. 1 showsmolecular weight markers that ranged from 500 bp to 1 kb (from MBIFermantas Inc.).

[0092] (3) Ultrastructural Morphology Changes in IPNV-Infected CHSE-214Cells Detected by Scanning Electron Microscopy

[0093] Apoptosis induces characteristic morphological changes in cells,such as condensation and fragmentation of the nucleus, as well as lossof cytoplasm. To substantiate further that IPNV-infected cells hadundergone nontypical apoptotic morphological changes such as membraneintegrity changed, negative control and IPNV-infected CHSE-214 cellswere harvested and processed for scanning electron microscopy as shownin FIG. 3. Negative control cells are shown in FIG. 3A. IPNV-infectedCHSE-214 cells at 8 h p.i. displayed detachment, cell rounding, andblebbing of membrane vesicles (MV) from the plasma membrane at thepre-late stage of apoptosis (20%; P<0.05), as shown in FIG. 3B.Middlelate-stage apoptotic cells (23%; P<0.05) are shown in FIG. 3C. Thecell membrane appears shrunken, and holes are present in the plasmamembrane. The hole sizes ranged from 0.39 to 0.78 μm with about 10 to 20holes per cell. A late-stage apoptotic cell (2%; P<0.05) is shown inFIG. 3D with the small holes still on the surface of the late-apoptoticcell.

[0094] (4) EGFP Release is Prevented by Protein Synthesis Inhibitor andTyrosine Kinase Inhibitor

[0095] In EGFP release experiments, EGFP was used to monitor theintegrity of the plasma membrane during apoptosis. As described in (3)above, small holes appeared in middle-late-stage apoptotic cells (FIG.3C). It is possible that intracellular material might leak out of thesesmall holes to the extracellular region before secondary necrosis. Theuse of EGFP to monitor the integrity of the plasma membrane ofIPNV-infected CHSE-214-EGFP cells is shown in FIG. 4. The EGFP releaseWestern blot assay result is shown in FIG. 4A, FIG. 4A, part a, showsthat the amount ot GFP decreased, especially between 8 and 16 h p.i. Theinternal control, actin protein, is shown in FIG. 4A, part b. Detectionof the EGFP released from the intracellular to the extracellular regionduring IPNV infection is shown in FIG. 4A, part c. The increase of GFPrelease began between 8 and 16 h p.i., which is consistent with FIG. 4A,part a. These data indicate that the membrane integrity changed quicklyat the middle-late apoptotic stage. The fluorometric EGFP release assayresults are shown in FIG. 4B. The open squares show that theintracellular amount of EGFP sharply decreased from 6 to 24 h p.i. butthat the largest release of EGFP occurred between 12 and 24 h p.i. Theopen diamonds show that the extracellular amount of EGFP increasedbetween 6 and 24 h p.i., which matches the intracellular data describedabove. EGFP was also used as a protein indicator to directly probemembrane integrity by immunoelectron microscopy. Normal CHSE-214-EGFPcells used as controls are shown in FIG. 5A. FIG. 5B shows that thesmall vesicle escaped from the membrane hole at the pre-late apoptoticcell stage and that the vesicle contains the same EGFP labeled by ananti-GFP polyclonal antibody-gold complex.

[0096] Drugs, including the protein synthesis inhibitor cycloheximide(CHX), the serine proteinase inhibitors aprotinin and leupeptin, thetyrosine kinase inhibitors genistein and tyrphostin, and the cationchelator EDTA, were used before IPNV infection to test the viability ofthese drugs on preventing membrane integrity change. Some of the drugs,such as CHX at 10 μg/ml and 2 mM EDTA, completely prevented EGFPrelease, and genistein at 100 μg/ml partially prevented EGFP release (asshown in FIG. 6A) to the extra-cellular region (as shown in FIG. 6C).The serine proteinase inhibitors aprotinin (400 μg/ml) and leupeptin(400 μg/ml) (FIG. 6C, lanes 5 and 6, respectively) and the tyrosinekinase inhibitor tyrphostin (100 μg/ml) (FIG. 6C, lane 8) did notprevent EGFP release. The internal control, actin protein, is shown inFIG. 6B for quantitation of protein loading per sample.

[0097] (C) Discussion

[0098] The present invention provides the first evidence that GFP can beused to sequentially monitor apoptotic morphological changes in livingcells. GFP is stable and species independent and can be monitorednoninvasively in living cells. However, working with GFP raisespractical problems. One such problem, common in fluorescence microscopyof live cells, is that of phototoxicity, which is thought to be causedmainly by fluorophore-mediated generation of free radicals. Fortunately,the introduction of mutant GFPs with higher quantum efficiencies,lower-energy excitation spectra, and better temperature stability hasbeen advantageous and has significantly widened the applicability of GFPto the study of proteins of low abundance.

[0099] For the purposes of studying the changes in membrane integrityafter IPNV infection, a clone with strong fluorescence intensity andnormal morphology, CHSE-214-EGFP, was selected and subcloned as a cellline for experiments as shown in FIG. 1. This clone uses a variant typeof GFP, EGFP, as a probe. The use of EGFP to study the morphologicalchanges during apoptosis clearly has advantages over GFP because EGFPproduces fluorescence 35 times more intense than GFP. The clones withlower fluorescence intensity did not produce a good image in sequentialmorphology studies.

[0100] The cloned CHSE-214-EGFP cells were expressed with EGFP (32.5kDa; as shown in FIG. 4A, lane 2), which is larger in molecular sizethan wild-type GFP (27 kDa; as shown in FIG. 4A, lane 1). But othercharacteristics of EGFP are similar to GFP. For example, GFP isfluorescent either as a monomer or as a dimer. The ratio of monomeric todimeric forms depends on the protein concentration and the environment.EGFP was also found in both control cells and IPNV-infected cells eitheras a monomer or as a dimer (as shown in FIG. 5). In fact, a doublet EGFPwas found in the released EGFP, as shown in FIG. 4A (part c, lane 6) and6C.

[0101] The results of using EGFP to monitor the dynamic morphologicalchanges in CHSE214-EGFP cells infected with IPNV are shown in FIG. 1.The series of events can be briefly divided into four stages: (i) theearly apoptotic stage (0 to 3 h p.i.), (ii) the middle apoptotic stage(3 to 6 h p.i.), (iii) the pre-late apoptotic stage (6 to 7 h p.i.), and(iv) the postapoptotic necrosis stage (after 7 h p.i.). Themorphological changes in apoptotic cells observed include celldetachment. rounding up, formation of MV, pinched off MV floating awayin the culture medium, and finally, postapoptotic necrosis.

[0102] The sequential morphological change events were different fromtypical apoptotic morphological changes, which are characterized asdetachment, rounding up, membrane blebbing, and finally the formation ofapoptotic bodies, as described by Wyllie et al. supra. The apoptoticprocess of CHSE-214 after IPNV infection is clearly defined as a“non-typical apoptotic morphological change” as summarized in FIG. 7,which include all of the typical characteristics of apoptosis, namely,DNA fragmentation, cell detachment and rounding up (FIGS. 7A-B),membrane blebbing and formation of membrane vesicles (MV) (FIG. 7C), andMV pinching off from the membrane which left small holes on the cellmembrane (FIG. 7D). However, at the late apoptotic stage, in addition tothe formation of apoptotic bodies (FIG. 7E) as in the typical apoptosis,the apoptotitc cells can undergo post-apoptotic necrosis (FIG. 7F) whichis characterized by the condensed chromatin enclosed by the nuclearmembrane.

[0103] Embodiment 2. Control of Cell Death via an Mcl-1 DependentPathway

[0104] Mcl-1 belongs to the Bcl-2 family, which is known as the“apoptosis-inhibiting protein”. The founding member of this family isthe bcl-2 protooncogene which was initially isolated from a follicularlymphoma (Bakhshi et al. (1985), Cell 41:889-906). Mcl-1 was originallyidentified from the differentiating human myeloid leukemia cell lineML-1. Its expression was found to increase early in the induction or“programming” of differentiation of ML-1 cells before the appearance ofdifferentiation markers. The coding region of mcl-1 was sequenced andfound to have a pronounced region of sequence homology to bcl-2 in thecarboxyl-terminal region (Kozopas et al. (1993), supra). Unlike bcl-2,mcl-1 contains a strong PEST sequence (enriched in proline, glutamicacid, serine and threonine) which is present in a variety of proteinsthat undergo rapid turnover. Overexpression of exogenously introducedmcl-1 has been shown to cause a prolongation of viability underconditions that normally cause apoptotic cell death, such as exposure tocytotoxic agents (e.g., the chemotherapeutic agent etoposide, calciumionophore, or UV irradiation) or the withdrawal of required growthfactors (Zhou et al (1997), Blood 89:630-643).

[0105] Although Mcl-1 has been studied in mammalian cells, no such studyhas been conducted in aquatic cells. In addition, the present inventionis the first to study the down regulation of Mcl-1 protein expression byviral infection. The experimental designs, results, and discussion ofthis embodiment are illustrated as follows:

[0106] (A) Experimental Design

[0107] (1). CHSE-214 and Viruses

[0108] Chinook salmon embryo cells (CHSE-214) and El-S of IPN virus Abstrain were prepared according to (1) of the Experimental Design inEmbodiment 1, supra.

[0109] (2). Scanning Electron Microscopy

[0110] The scanning electron microscopy was prepared according to (5) ofthe Experimental Design of Embodiment 1, supra.

[0111] (3). DNA Preparation and Gel Electrophoresis

[0112] The DNA preparation and agarose gel electrophoresis were preparedaccording to (4) of the Experimental Design of the Embodiment 1, supra.

[0113] (4). Immunoblotting

[0114] About 10⁵ cells/ml were seeded on a 60-mm. petri dish for growthfor more than 20 h. Monolayers were rinsed twice with phosphate-bufferedsaline (PBS). Control cells or cells that received virus at a MOI of 1were incubated for 0, 2, 4, 6, 8, 10 and 24 h. At the end of eachincubation time the culture medium was aspirated. The cells were washedwith PBS and then lysed in 0.3 nil lysis buffer [10 mM Tris base, 20%glycerol, 10 mM sodium dodecyl sulfate (SDS), 2% 13-mercaptoethanol(P-ME), pH 6.8].

[0115] Proteins were separated by SDS-polyacrylamide gel electrophoresis(Laemmli, 1970), electroblotted, and subjected to immunodetection asdescribed by Kain et al. (1994). Blots were incubated with a 1:1500dilution of anti-human Mcl-1 polyclonal antibodies (Pharmingen) or a1:7500 dilution of a peroxidase-labeled goat anti-rabbit conjugate(Amershan). Chemi-luminescent detection was performed according to theinstructions provided with the Western Exposure ChemiluminescentDectection System (Amershan). Chemiluminescent signals were imaged byexposure to Kodak XAR-5 film (Eastman Kodak, Rochester, N.Y., USA).Primary (Mcl-1) and secondary antibodies (peroxidase-labeled goatanti-rabbit conjugate) were stripped from blots by incubation instripping buffer containing 62.5 mM Tris-HCI (pH 6.8), 3.0% (w/v) SDSand 50 mM 1,4-dithiothreitol for 30 min at 55° C. with gentle shaking.The blots were then washed four times for 10 min each time in PBScontaining 0.1% (v/v) Tween 20 and reprobed with mouse actin monoclonalantibody (1/1500, Chemicon) and a 1:7500 dilution of aperoxidase-labeled sheep anti-mouse conjugate (Amersham).

[0116] The potent drugs on effect of blockage on viral proteinexpression experiment, the cell preparation was as described aboveexcept that extra, cycloheximide (10 μg4 g/ml), aprotinin (400 μg/ml),leupeptin (400 μg/ml), genistein (100 μg/ml), tryphostin (100 μg/ml) andEDTA (2 mM) were added to 3 nil of MEM medium on CHSE-214 cells beforevirus infection and incubation for 16 h. At the end of the incubationperiod, cells were harvested and the samples were analyzed by Westernblot method.

[0117] (B) Results

[0118] (1). Ultrastructure Morphological Changes in CHSE-214 Cells withIPNV Infection by Scanning Electron Microscopy

[0119] Apoptosis induces characteristic morphological changes in cells,such as condensation and fragmentation of the nucleus as well as loss ofcytoplasm (Wyllie et al. (1984), supra). To substantiate thatIPNV-infected cells had undergone apoptosis, negative control andIPNV-infected cells were harvested and processed for scanning electronmicroscopy. Normal negative control cells are shown in FIG. 8A.IPNV-infected CHSE-214 cells displaying detachment and blebbing of theplasma membrane are shown in FIG. 8B.

[0120] (2). Induction of Internucleosomal Cleavage by IPNV in CHSE-214Cells

[0121] DNA fragmentation is a well-defined biochemical marker ofapoptosis. El-S of IPN virus Ab strain (MOI of 1) infected cells wereexamined for evidence of internucleosomal fragmentation. Intenseinternucleosomal fragmentation of DNA, a pattern highly specific toapoptosis, was observed in CHSE-214 cells infected with IPNV (FIG. 9).The IPNV induced DNA fragmentation at 8 h and 12 h postinfection wasidentified by gel electrophoresis (FIG. 9, lanes 4 and 5). The gel ofthe negative control at 0 h incubation and 4 h postinfection showed noDNA fragmentation (FIG. 9, lanes 2 and 3).

[0122] (3). Western Blot

[0123] The characterization of the viral protein size and Mcl-1expression was directly quantified by Western blots from CHSE-214 cells.FIG. 10 shows the major protein expression pattern during infection ofCHSE-214 cells by a MOI of 1 of El-S. The viral proteins had a largeexpression after 4 h post-infection. FIG. 11 shows the Mcl-1 proteinexpression pattern during infection of CHSE-214 cell with a MOI of 1 ofEl-S. The down-regulation of Mcl-1 expression occurred between 6 h and 8h post-infection (as shown in FIG. 11A, lanes 4 and 5). The internalcontrol actin is shown in FIG. 10B.

[0124] (4) Blocking of Virus Replication for Prevention ofDown-Regulation of the Mcl-1 Protein by Certain Drugs

[0125] To confirm whether viral replication is involved indown-regulation of Mcl-1, viral replication in host cell was blocked bytreatment with certain drugs. When the protein synthesis inhibitors, 10μg/ml cyclohexamide, tyrosine kinase inhibitor, 100 μg/ml of genisteinor the cation chelator, 2 mM of EDTA, were added to CHSE-214 cellsbefore IPNV infection, viral replication was prevented (as shown in FIG.12A). At the same time, these same drugs partially preventeddown-regulation of Mcl-1 protein expression (as shown in FIG. 12B).However, the serine proteinase inhibitor aprotinin 400 μg/ml andleupeptin 400 μg/ml (as shown in FIG. 12B, lanes 5-6) and the tyrosinekinase inhibitor tryphostin 100 μg/ml (as shown in FIG. 12B, lane 8)could not. The internal control actin protein is shown in FIG. 12C. TheDNA internucleosomes were assayed under the same conditions describedabove. The blocking of viral replication groups consistently andstrongly prevented the induction of internucleosomal cleavage by IPNV(as shown in FIG. 13), with the exception of the 2 mM EDTA treatmentgroup which displayed minor internucleosomal cleavage (as shown in FIG.13, lanes 14 and 15).

[0126] (5). Intact Cell System

[0127] In order to determine whether IPNV uses the VP3 protein intriggering host cell apoptosis, we designed different sized VP3antisense RNA molecules (i.e., A200, A400, and A700) and inserted theminto a blank plasmid pcDNA3 to form pA200 (nt. 501-711), pA400 (nt.285-711) and pA700 (nt. 1-711) plasmids. These and blank insert pcDNA3(Invitrogen, U.S.A.) and used selection with G418 to generate stablecell lines. The stable cells were then infected with IPNV (MOI 1). Atthe end of incubation time, the cell survival rate was assayed by use ofthe trypane blue method (results shown in FIG. 14 [phase-contrastimages] and FIG. 15). In particular, at 24 hours post-incubation (p.i.),it was found that pA200 could increase survival up to 40%. VP3 antisenseRNA molecules pA200, pA400 and pA700 were prepared using VP3 specificprimers P1, P2, P3, and P4 for amplification. Specifically, VP3specificprimers P1 and P2 were used to amplify and generate A200, whichcontained base No. 501 to 711 of the VP3 antisense RNA, by PCR using taqpolymerase; VP3-specific primers P1 and P3 were used to amplify andgenerate A400, which contained base No. 285 to 711 of the VP3 antisenseRNA; VP3-specific primers P1 and P4 were used to amplify and generateA700, which contained base No. 1 to 711 of the VP3 antisense RNA. TheA200, A400, and A700 VP3 antisense RNA molecules were constructed topcDNA3.11V5His-TOPO vector as pA200, pA400, and pA700, respectively.

[0128] (6). VP3 Gene Assay in Different Cell Lines

[0129] The fused gene, C1-EGFP-VP3, was inserted into a plasmid.pEGFP-C1 is a commercially available vector which contains a strongpromoter (CMV promoter) and the EGFP gene. To produce the fused gene,VP3 gene was amplified using VP3-specific primers P1 and P2 by PCR andligated to a pcDNA3.1 vector. The inserted VP3 in pcDNA3. 1 vector wasthen cut with restriction enzymes Hind III and EcoRI, and re-constructedto a pEGFP-C 1 vector from the HindIII and EcoRI sites. Individual genefunction was tested in several different cell lines, including fishCHSE-214, mammalian cells NIH3T3 (Rat) and CHO (Hamster) cells and tumorcell as Hepa-3b and Hepa-G2 (Human). The EGFP-VP3 was transfected tocell lines with lipofectamine. Lipofectamine resembles a liposome whichhas the capability of forming a lipid bilayer to assist DNAtransfection. VP3 induced NIH3T3 cell apoptosis (as shown in FIG. 16),as indicated by the percentage of positive apoptotic cells (as shown inFIG. 17) that were induced (about 20% at 36 hours p.i.). Apoptosis couldbe similarly induced by VP3 in other cell lines.

[0130] (7). VP3 Induced Zebrafish Embryonic Cell Death and Suppressionby VP3 Antisense RNA

[0131] Using zebrafish embryos as an in vivo assay system, one or twocell stage embryos were transfected with about 20 pg of VP3 usingmicroinjection. (George Streisinger, “Zebrafish”, University of OregonPress, Edition 2.1, (1994)).

[0132] DNA fragmentation was analyzed in zebrafish embryonic cellsinjected with VP3. At end of the incubation time, the genomic DNA fromthe live and dead embryos was isolated and analyzed in 1.2% agarose gel(as shown in FIG. 18). It was found that VP3 induced embryo cell deathby increasing DNA laddering (as shown in Lane 6 of FIG. 18), as comparedto live embryos (as shown in Lanes 3 and 6 of FIG. 18) and withpEGFP-VP3 microinjection for 4 hours (as shown in Lane 5 of FIG. 18).

[0133] Overexpression of the IPNV-VP3 induced embryonic cell death. Alsoshown is the micrographs of zebrafish embryos where apoptosis wastriggered by microinjection with pEGFP-VP3 (as shown in FIGS. 19(C) &D), as contrasting to embryos microinjected with pEGFP-C1, the negativecontrol (FIGS. 19(A) & (B)). Also, as shown in FIG. 19D, flattened deadcells and damaged somite appeared in the embryo microinjected withpEGFP-VP3 for 24 hours, probably due to overexpression of VP3. Thedamaged somite was more severe after 48 hours of microinjection withpEGFP-VP3 into zebrafish embryos, as seen in FIG. 20D.

[0134] Interestingly, when antisense RNA of VP3 was provided togetherwith pEGFP-VP3, the % of alive embryos after microinjection withpEGFP-VP3 increased proportionally, as shown in FIG. 21, demonstratingthat antisense VP3 RNA has the capability of overcoming cell deathcaused by overexpression of VP3. The same phenomenon was furthersupported by FIG. 21, where the % of defected embryos after 60 hours ofmicroinjection with pEGFPVP3 and pEGFP-VP3 plus antisense VP3 RNA wasmeasured. In the group where pEGFPVP3 plus antisense VP3 RNA wasmicroinjected, the % of defected embryos was about 13%, contrasting toabout 29% of defected embryos in the group where pEGFP-VP3 wasmicroinjected. Further evidence showing that the leakage of defectedzebrafish embryos was rescued by the addition of antisense VP3 RNA isprovided in FIG. 23C.

[0135] (8). Bcl-2 Family Member, zfMcl-1a Block VP3 Death Function

[0136] zfMcl-1a and zfBcl-xL were cloned by Chen et al (Chen M.C., GongH. Y., Cheng C. Y., Wang J. P, Hong, J. R. and Wu J. L. (2000) Biochem.Biophys Res. Com. 279, 725-731). In order to test whether this proteincould block VP3 function in zebrafish embryo, VP3 was microinjectedalong with zfMcl-1a or zfBcl-xL (1:1=20 pg:20 pg). The protocol formicroinjection of VP3 and zfmcl-1a plasmid DNA was according to Chapter5, George Streisinger, “Zebrafish”, University of Orgon Press, Edition2.1, (1994), which is herein incorporated by reference.

[0137] The results (FIG. 24 and 25), which were determined after 36hours of microinjection, demonstrate that zfMcl-1a enhanced embryonicsurvival by about 38% (FIG. 25, columns 3 and 4) and decreased embryonicdefects about 20% (FIG. 24, columns 3 and 4).

[0138] (C) Discussion

[0139] IPNV is a highly contagious disease of susceptiblehatchery-reared trout and Japanese eel in Taiwan. IPNV replicates in avariety of continuous cell lines from teleost fish at temperatures below24° C. The virus replicates in the cytoplasm and a single cycle ofreplication takes 16-20 h at 22° C. resulting in a characteristiccytopathic effect (CPE). Rainbow trout gonad (RTG-2) cells infected withIPNV yielded infectious titers of only 10⁶-10⁷ pfu/Ml, because the RTG-2cells can produce interferon when IPNV infected and are themselvessensitive to interferon treatment, as reported by MacDonald and Kennedy(Virology (1979), 95:260-264). On the other hand, chinook salmon embryo(CHSE-214) cells do not produce interferon, and are insensitive tointerferon treatment, resulting in virus yields of 2-5×10⁸ pfu/ml orhigher.

[0140] In the present invention, by assaying for cellultra-morphological features (as shown in FIG. 8) and DNA fragmentation(as shown in FIG. 9), demonstrates that apoptosis contributed to thedeath of IPNV infected cells.

[0141] Also, viral replication (as shown in FIG. 10) correlates withdown-regulation of Mcl-1 protein expression (as shown in FIG. 11A). Thisis the first study that confirms down-regulation of Mcl-1 (a member ofthe Bcl-2 family) protein expression by viral infection.

[0142] In view of the capacity of Mcl-1 to block or delay apoptosis andits sequence feature as a PEST (proline, glutamic acid, serine,threonine) protein that can be degraded rapidly, one possible role ofthis protein is as a rapid turnover effector that controls the rate ofapoptosis. However, when treatment with some drugs before IPNV-infectedCHSE-214 cells was performed, the protein synthesis inhibitor,cycloheximide, or the tyrosine kinase inhibitor, genistein, and thecation chelator, EDTA, all blocked viral protein expression (as shown inFIG. 12A). And at the same time, the same drugs helped to maintain Mcl-1expression level (as shown in FIG. 12) and blocked the induction of DNAintemucleosomal cleavage (as shown in FIG. 13) for rescue or delay ofapoptotic cell death.

[0143] VP3, a 32-kDa protein derived from the IPNV segment A, inducesapoptosis in fish CHSE-214 and mammalian cells, including the NIH3T3(Mouse) and CHO (Hamster) cells and tumor cell as Hepa-3b and Hepa-G2(Human). The results presented herein above demonstrate that VP3 can besuppressed by VP3 antisense RNA thereby enhancing the survival rateduring IPNV infection. On the other hand, the VP3 expression alone caninduce zebrafish embryonic cell death and DNA fragmentation. Deathcaused by VP3 can be prevented by anti-apoptosis protein zfMcl-1a, aBcl-2 family member from zebrafish. It appears that VP3 is a functionaldeath gene in late IPNV replication cycle that the effect of VP3 can beblocked by VP3 antisense RNA or functionally blocked by anti-apoptoticgene zfmcl-1a.

[0144] Having described the invention in detail and by reference to thepreferred embodiments it will be apparent to those skilled in the artthat modifications and variations are possible without departing fromthe scope of the invention as defined in the following appended claims.

We claim:
 1. An aquatic apoptotic cell comprising: an aquatic cell,wherein said aquatic cell is infected with an aquabimavirus, whereinsaid aquabimavirus is an infectious pancreatic necrosis virus (IPNV). 2.The aquatic apoptotic cell according to claim 1, wherein said aquaticcell is detected by transfecting said aquatic cell with a pEGFP-N1vector; and monitoring morphological changes by a fluorescencemicroscopic technique.
 3. The aquatic apoptotic cell according to claim1, wherein said aquatic cell is a fish cell which is selected from thegroup consisting of rainbow trout (Oncorhynchus mykiss), brook trout(Salvelinus fontinolis), chinook salmon (Oncorhynchus tshawytscha), cohosalmon (Oncorhynchus kisutch), sockeye salmon (Oncorhynchus nerca),Atlantic salmon (Salmo salar), carp, perch, pike, eels, zebrafish andchar.
 4. A method for detecting an aquatic apoptotic cell according toclaim 1 comprising: transfecting an aquatic cell with a pEGFP-N1 vector;infecting said aquatic cell with an aquabirnavirus; and monitoringmorphological changes by a microscopic technique.
 5. The methodaccording to claim 4, wherein said microscopic technique is at least oneselected from the group consisting of light microscopy, fluorescencemicroscopy, scanning electron microscopy, and immunoelectron microscopy.6. A method for detecting an aquatic apoptotic cell according to claim 1comprising: transfecting an aquatic cell with a pEGFP-N1 vector toproduce EGFP in said aquatic cell; infecting said aquatic cell withIPNV; and measuring said EGFP in said aquatic cells.
 7. A method forpreventing apoptosis in an aquatic cell which is caused by infectionwith infectious pancreatic necrosis virus (IPNV) comprising: treatingsaid aquatic organism with one selected from the group consisting ofcyclohexamide, genistein and EDTA prior to infection with saidaquabimavirus.
 8. The method according to claim 7, wherein said aquaticcell is a fish cell, which is selected from the group consisting ofrainbow trout (Oncorhynchus mykiss), brook trout (Salvelinusfontinolis), chinook salmon (Oncorhynchus tshawytscha), coho salmon(Oncorhynchus kisutch), sockeye salmon (Oncorhynchus nerca), Atlanticsalmon (Salmo salar), carp, perch, pike, eels and char.
 9. A method forinducing apoptosis in a host comprising: transfecting said host aplasmid containing a VP3 gene.
 10. The method according to claim 9,wherein said host is an aquatic or a vertebrate selected from the groupconsisting of rainbow trout (Oncorhynchus mykiss), brook trout(Salvelinus fontinolis), chinook salmon (Oncorhynchus tshawytscha), cohosalmon (Oncorhynchus kisutch), sockeye salmon (Oncorhynchus nerca),Atlantic salmon (Salmo salar), carp, perch, pike, eels, char, mouse,hamster and human.
 12. An agent for inducing apoptosis comprising aneffective amount of VP3 protein.
 13. A method for preventing or rescuingapoptosis in a host comprising: transfecting said host an antisense VP3RNA or a zfMcl-1a gene.
 14. The method according to claim 13, whereinsaid host is an aquatic or a vertebrate selected from the groupconsisting of rainbow trout (Oncorhynchus mykiss), brook trout(Salvelinus fontinolis), chinook salmon (Oncorhynchus tshawytscha), cohosalmon (Oncorhynchus kisutch), sockeye salmon (Oncorhynchus nerca),Atlantic salmon (Salmo salar), carp, perch, pike, eels and char,zebrafish, mousr, hamster and human
 15. The method according to claim14, wherein said host is an embryo of said aquatic or vertebrate. 16.The method according to claim 13, wherein said apoptosis is caused byIPNV infection.
 17. The method according to claim 13, wherein saidapoptosis is caused by VP3 transfection.
 18. The method according toclaim 13, wherein host is a cell line which is one selected from thegroup consisting of CHSE, NIH3T3, and CHO.
 19. An agent for preventingor rescuing apoptosis comprising an effective amount of VP3 antisenseRNA.
 20. An agent for preventing or rescuing apoptosis comprising aneffective amount of zfMcl-1a potein.